Using T cell receptor (TCR)-transgenic (N15 tg) beta2-microglobulin deficient RAG2-/-H-2b mice specific for the VSV8 (RGYVYQGL) octapeptide bound to K/b, we observed that a single weak agonist peptide variant of VSV8 (V4L), termed LM, induces T cell maturation while the cognitive VSV8 peptide, in contrast, triggers negative selection. To characterize the molecular basis of this observation, a crystal structure of the L4/kappaB complex was refined to 2.1A for comparison with the VSV8/kappaB complex at a similar resolution. Aside from the methylene extension at the p4 position of L4 and resulting alteration of the exposed KbLys66 side chain, these structures are essentially identical yet result in very different selection outcomes. To now characterize the natural peptides involved in positive selection in vivo, four aims will be pursued. First, mass spectrometry approaches will be used to identify thymic Kb binding peptides which select the N15 receptor in N15tgRAG2-/-beta2M- /-. Subsequently, exposure of thymic cultures to mAbs specific for a given peptide/MHC (pMHC) complex will allow us to ascertain whether there is an obligate require for a single peptide in the positive selection process. If so, the gene encoding the predominant naturally selecting peptide will be disrupted. The effects of these natural peptides on selection of other VSV8/Kb-specific TCRs including N26 will be determined. Second, we will produce and utilize peptide/Kb tetramers to display selected and post-selected thymocyte populations. The molecular features of the TCRs which interact with VSV8/Kb will be determined using single cell PCR-based methodologies. Assuming several natural peptides are capable of positively selecting VSV8/Kb specific T cells, comparison of the individual repertoires elicited will be made. Third, x- ray crystallographic techniques will be used to determine the structural basis of focal differences in the various positively selecting native pMHC complexes. Common features will be identified including any peptide induced alterations of exposed MHC alpha-helical side chains. Fourth, the potential role for germline Valpha genes in positive selection will be assessed, with emphasis on CDR1 as suggested by x-ray analysis of N15- VSV8/K/b co-crystals.